Heterologous NH2-terminal basolateral targeting sequence for multipass transmembrane protein in epithelial cells

Sodium-dependent vitamin C transporters (SVCTs), SVCT1 and SVCT2, share 66% identical amino acid sequence but differentially localize to the apical and basolateral surface in MDCK cells, respectively. The N-terminal sequence of SVCT2 plays a crucial rule for basolateral targeting and can redirect the apically localized SVCT1 to the basolateral membrane after N-terminus replacement. This effect also been reported for three other pairs of multipass transmembrane proteins. To determine whether the N-terminal basoloateral targeting signals (BTS) are universal among the multipass membrane proteins, heterologous putative N-terminal BTS insertions and C-terminal deletion mutations were constructed in the EGFP- tagged human SVCT1M31m plasmid that was missing the first N-terminal 30 amino acids and had been shown previously to localized in the apical membrane. Mutant proteins stably expressed in the MDCK cells were analyzed by live cell confocal imaging, functional assays, and quantification of fluorescence to assess the effects of the insertion and deletion mutations. Putative AQP3 and NaDC3 N-terminal BTS insertion reduced apical membrane incorporation or retention of the hSVCT1 protein, whereas surprisingly, the insertion of the putative NET BTS had no effect on the apical sorted hSVCT1. These results suggested that the putative BTS insertions tested in this study did affect the apical localization of hSVCT1. However, they were unable to redirect the protein to the basolateral membrane. Moreover, the two termini of the protein maybe are both needed to cooperate with each other to fold correctly to be targeted to the basolateral membrane.